The serotonin receptor subtype 2C (5-HT2CR) is a G protein-coupled receptor that plays an important role in feeding behavior and tonic inhibition of the neuronal network excitability. The expression of the 5-HT2CR is detected only in the central nervous system (CNS) and is regulated during brain development. To understand the regulation, function and potential disease association of the S-HT2CR, we isolated the complete human 5HT2CR cDNA and characterized its gene structure and 5' flanking sequence. In addition to the normal 4.8-kb 5HT2CR cDNA, we identified two other cDNA variants; one is derived from an alternatively spliced RNA and the other from the S-HT2CR transcript which undergoes mRNA editing. Also, we found that the 5' flanking 5Hl about2CR sequence can confer cell type-specific expression and direct transgene expression to the brain. Several positive and negative regulatory regions have been identified. In particular, we have located a negative regulatory region which appears to cooperate with multiple upstream elements to regulate 5-HT2CR promoter expression. In this application, we propose to further investigate the regulation of 5-HT2CR gene. The central hypothesis of this proposed research is that that the negative regulatory element is important for proper temporal and spatial expression of the 5-HT2CR in the CNS. In support of this hypothesis, two sets of molecular and developmental experiments combining transgenic approach with in vitro studies are proposed. First, we plan to characterize the negative regulatory element and its binding protein important for S-Hl2CR expression. We will first confirm whether the novel negative element regulates 5-HT2CR promoter expression at the level of transcription. We will define the precise location of the negative regulatory element and attempt to identify the negative element-binding protein. Second, we will study the neural-specific and developmental expression of the human 5-HT2CR promoter and examine if the negative regulatory element is important for its expression pattern during development. We plan to generate transgenic mice carrying the beta-galactosidase gene driven by the 5HT2CR promoter with or without the negative regulatory sequence. We will analyze the transgene expression pattern in the CNS and compare to that of the endogenous 5-HT2CR gene. Upon these analyses, I hope to provide a better understanding of the neural specificity of this important neurotransmitter receptor expression, ultimately leading to a thorough understanding of the complex communication mechanisms within the CNS including the choroid plexus, a important tissue for the blood-brain barrier.